Native Myeloperoxidase Research Articles

The role and mechanism of myeloperoxidase in dermatomyositis

ObjectiveDermatomyositis (DM) is the best known subtype of idiopathic inflammatory myopathies. The hallmarks of DM muscle pathology including microangiopathy, inflammatory infiltration, and perifascicular atrophy. Recent findings have revealed pathogenetic effects of myeloperoxidase (MPO) by causing oxidative damage and regulating abnormal immunity in multiple disease conditions. In this study, we aimed to explore the role of MPO in the pathogenesis of DM. MethodsThe peripheral blood mononuclear cell (PBMC) mRNA expression and DNA methylation of MPO were verified using real-time qPCR and bisulfite pyrosequencing, respectively. Plasma MPO levels were measured with enzyme-linked immunosorbent assay, and their relationships with clinical characteristics were analyzed. The expression and distribution of MPO in muscle were tested by immunofluorescence. Purified human native MPO protein was used to stimulate human dermal microvascular endothelial cells (HDMECs) and skeletal muscle myotubes. The cell viability, tube forming capacity, permeability, adhesion molecule expressions in HDMECs, and atrophy and programmed cell death pathways in myotubes were then observed. ResultsMPO gene methylation was decreased, while mRNA expression and plasma levels were increased in DM. Plasma MPO of DM patients was positively correlated with serum creatine kinase (CK). MPO mainly distributed around endomysia capillaries and perifascicular atrophy in DM muscle biopsies, and was co-localized with CD4+, CD8+ T cells and CD19+ B cells. MPO not only could influence the cell viability, tube forming capacity, permeability and expression of adhesion molecules (including ICAM 1, VCAM 1 and E-selectin) of HDMECs, but also could cause atrophy of myotubes. ConclusionsOur study disclosed, for the first time, that MPO plays an important role in promoting inflammatory infiltration and inducing muscle damage in DM patients. MPO may be a potential biomarker for DM muscle involvement and MPO targeted drugs may be promising in DM treatment.

Structure-biological activity relationships of myeloperoxidase to effect on platelet activation

Myeloperoxidase (MPO), an oxidant-producing enzyme of neutrophils, has been shown to prime platelet activity promoting immunothrombosis. Native MPO is a homodimer, consisting of two identical protomers (monomer) connected by a single disulfide bond. But in inflammatory foci, MPO can be found both in the form of a monomer and in the form of a dimer. Beside MPO can also be in complexes with other molecules and be modified by oxidants, which ultimately affect its physicochemical properties and functions. Here we compared the effects of various forms of MPO as well as MPO in complex with ceruloplasmin (CP), a physiological inhibitor of MPO, on the platelet activity. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. MPO was modified with HOCl in a molar ratio of 1:100 (MPO-HOCl). Using surface-enhanced Raman scattering (SERS) spectroscopy we showed that peaks at about 510 and 526 cm−1 corresponded to disulfide bond was recognizable in the SERS-spectra of dimeric MPO, absent in the spectrum of hemi-MPO and less intense in the spectra of MPO-HOCl, which indicates the partial decomposition of dimeric MPO with a disulfide bond cleavage under the HOCl modification. It was shown hemi-MPO to a lesser extent than dimeric MPO bound to platelets and enhanced their agonist-induced aggregation and platelet-neutrophil aggregate formation. MPO modified by HOCl and MPO in complex with CP did not bind to platelets and have no effect on platelet activity. Thus, the modification of MPO by HOCl, its presence in monomeric form as well as in complex with CP reduces MPO effect on platelet function and consequently decreases the risk of thrombosis in inflammatory foci.

Recombinant Myeloperoxidase as a New Class of Antimicrobial Agents.

ABSTRACTHeme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. The difficulty of producing MPO at a large scale hinders its study and utilization. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study. rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities. In this study, rMPO was secreted into culture medium as a monomer. rMPO revealed enzymatic activity similar to that of native MPO. rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. In animal safety tests, the administration of 100 nM rMPO via tail vein did not result in any sign of toxic effects. Taken together, the data suggest that rMPO purified from a stably expressing human cell line is a new class of antimicrobial agents with the ability to kill a broad spectrum of pathogens, including bacteria and fungi with or without drug resistance.IMPORTANCE Over the past 2 decades, more than 20 new infectious diseases have emerged. Unfortunately, novel antimicrobial therapeutics are discovered at much lower rates. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death, and high health care costs. Currently, this is best seen with the lack of a cure for coronavirus disease 2019 (COVID-19). To combat such untreatable microorganisms, there is an urgent need to discover new classes of antimicrobial agents. Myeloperoxidase (MPO) plays an important role in host defense. The difficulty of producing MPO on a large scale hinders its study and utilization. We have produced recombinant MPO at a large scale and have characterized its antimicrobial activities. Most importantly, recombinant MPO significantly reduced the morbidity and mortality of murine pneumonia induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. Our data suggest that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 μM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV–Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 μM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100–200 μM). In contrast, low concentrations of edaravone (1–10 μM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0–10 μM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Identification and characterization of myeloperoxidase in orange-spotted grouper (Epinephelus coioides)

Cryptocaryon irritans is an important protozoan ciliate, which has led to heavy economic losses in marine aquaculture. Previous studies have indicated that C. irritans infection could induce the migration of neutrophils to infection sites. Myeloperoxidase (MPO) mainly exists in the cytoplasmic granules of the neutrophil and performs its function by a unique enzymatic capacity to produce hypohalous acid and other toxic oxidants. To determine the involvement of MPO and neutrophils against C. irritans infection in the host, we amplified MPO cDNA (EcMPO) from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcMPO encodes a putative polypeptide of 770 amino acids and has typical structural characteristics of mammalian MPO, including a signal peptide, a propeptide, a light chain, a heavy chain, and a peroxidase domain. Bioinformatics analysis has demonstrated that the most important functional sites in mammalian MPO were also conserved in grouper and other piscine MPO, implying the functional conservation of this protein during evolution. A rabbit anti-MPO recombinant protein polyclonal antibody was produced, which could recognize the native MPO protein. The expression of EcMPO was higher in the lympho-hematopoietic organs, such as head kidney, trunk kidney, spleen, but lower in muscle, heart, and brain. After infection with C. irritans, the EcMPO transcript was significantly up-regulated at specific time points in the infection sites (skin and gill) and systemic immune organs (head kidney and spleen); The number of EcMPO positive cells first increased and then decreased in the gill, but was still higher than the control after 7 days. These results demonstrated that EcMPO and its positive cells may be involved in anti-C. irritans infection in the grouper, which is attributed to the innate immune mechanisms of the host against parasite infection.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca2+, and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

Clinical features of patients with anti-neutrophil cytoplasmic autoantibodies targeting native myeloperoxidase antigen

Objectives Anti-neutrophil cytoplasmic autoantibodies (ANCA) are useful diagnostic markers in systemic vasculitic disorders with small-vessel involvement, but depending on the particular test used, the myeloperoxidase (MPO)-ANCA results are variable. In the present study, we performed a comparative analysis between our originally developed nMPO-ANCA assay that targets the native MPO antigen and other commercially available assays using sera of patients with clinical features of ANCA-associated vasculitis (AAV).Methods Sera of 24 patients strongly suspected of having AAV were examined for the presence of MPO-ANCAs by our nMPO-ANCA assay and by other commercial-based MPO-ANCA assays. These results were correlated to indirect immunofluorescence microscopy staining patterns and patient clinical parameters.Results Eighteen out of 24 patients (75 %) were positive for nMPO-ANCA, compared with 13 out of 24 patients (54 %) by one of the most frequently used commercial-based MPO-ANCA enzyme-linked immunosorbent assays (ELISAs) in Japan. Interestingly, the patients who tested positive with our nMPO-ANCA assay alone showed clinical features of AAV marked by continuous fever, polyarthritis, and mild nephritis. The titers of nMPO-ANCA decreased in association with clinical improvement after treatment.Conclusions Our data suggest that a positive nMPO-ANCA result, which identifies antibodies to human native MPO antigen, correlates with AAV disease activity. Moreover, the nMPO-ANCA test has clinical utility in detecting AAV-affected patients who have tested negative using commercially available assays.

Genetically Determined Severity of Anti-Myeloperoxidase Glomerulonephritis

Myeloperoxidase (MPO) is a target antigen for antineutrophil cytoplasmic autoantibodies (ANCA). There is evidence that MPO-ANCA cause necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. NCGN severity varies among patients with ANCA disease, and genetic factors influence disease severity. The role of genetics in MPO-ANCA NCGN severity was investigated using 13 inbred mouse strains, F1 and F2 hybrids, bone marrow chimeras, and neutrophil function assays. Mouse strains include founders of the Collaborative Cross. Intravenous injection of anti-MPO IgG induced glomerular crescents in >60% of glomeruli in 129S6/SvEv and CAST/EiJ mice, but <1% in A/J, DBA/1J, DBA/2J, NOD/LtJ, and PWK/PhJ mice. C57BL6J, 129S1/SvImJ, LP/J, WSB/EiJ, NZO/HILtJ, and C3H mice had intermediate severity. High-density genotypes at 542,190 single nucleotide polymorphisms were used to identify candidate loci for disease severity by identifying genomic regions that are different between 129S6/SvEv and 129S1/SvImJ mice, which are genetically similar but phenotypically distinct. C57BL/6 × 129S6 F2 mice were genotyped at 76 SNPs to capture quantitative trait loci for disease severity. The absence of a dominant quantitative trait locus suggests that differences in severity are the result of multiple gene interactions. Invivo studies using bone marrow chimeric mice and invitro studies of neutrophil activation by anti-MPO IgG indicated that severity of NCGN is mediated by genetically determined differences in the function of neutrophils.

Clinical features of patients with anti-neutrophil cytoplasmic autoantibodies targeting native myeloperoxidase antigen

Anti-neutrophil cytoplasmic autoantibodies (ANCA) are useful diagnostic markers in systemic vasculitic disorders with small-vessel involvement, but depending on the particular test used, the myeloperoxidase (MPO)-ANCA results are variable. In the present study, we performed a comparative analysis between our originally developed nMPO-ANCA assay that targets the native MPO antigen and other commercially available assays using sera of patients with clinical features of ANCA-associated vasculitis (AAV). Sera of 24 patients strongly suspected of having AAV were examined for the presence of MPO-ANCAs by our nMPO-ANCA assay and by other commercial-based MPO-ANCA assays. These results were correlated to indirect immunofluorescence microscopy staining patterns and patient clinical parameters. Eighteen out of 24 patients (75%) were positive for nMPO-ANCA, compared with 13 out of 24 patients (54%) by one of the most frequently used commercial-based MPO-ANCA enzyme-linked immunosorbent assays (ELISAs) in Japan. Interestingly, the patients who tested positive with our nMPO-ANCA assay alone showed clinical features of AAV marked by continuous fever, polyarthritis, and mild nephritis. The titers of nMPO-ANCA decreased in association with clinical improvement after treatment. Our data suggest that a positive nMPO-ANCA result, which identifies antibodies to human native MPO antigen, correlates with AAV disease activity. Moreover, the nMPO-ANCA test has clinical utility in detecting AAV-affected patients who have tested negative using commercially available assays.

A predictive kinetic model for inhibitory effect of nitrite on myeloperoxidase catalytic activity towards oxidation of chloride

Myeloperoxidase (MPO) is a neutrophil enzyme that employs hydrogen peroxide (H2O2) to catalyze the oxidation of chloride (Cl–) to hypochlorous acid (HOCl). Accepted mechanism is based on rapid reaction of native MPO with H2O2to produce Compound I (MPO-I) which oxidizes Cl– through a 2e– transition generating MPO and HOCl. MPO-I also reacts with H2O2 to generate Compound II (MPO-II) which is inactive in 2e oxidation of Cl–. Nitrite ( NO2-) inhibits the 2e oxidation of Cl– by reaction with MPO-I through 1e transition generating MPO-II and nitrite radical. H2O2 consumption during steady- state catalysis was monitored amperometrically by a carbon fiber based H2O2-biosensor at 25oC. Results demonstrated that in absence of NO2- reactions were monophasic and rapid (complete H2O2 consumption occurs in 2- increases, reactions change to biphasic (rapid step followed by a slow step) and both steps have been inhibited by NO2- . A predictive kinetic model describing the inhibittory effect of NO2- was developed and applied to experimental results The model is based on the assumption that MPO–I cannot be detected during steady-state catalysis. Calculated rate constants are in agreement with those obtained from pre-steady state kinetic methods.

Partially Hydrolyzed Guar Gum Attenuates the Severity of Pouchitis in a Rat Model of Ileal J Pouch-Anal Anastomosis

We aimed to evaluate the efficacy of treatment with partially hydrolyzed guar gum (PHGG) using a rat model of ileal pouch-anal anastomosis and pouchitis. In the J pouch groups, tissue myeloperoxidase activities were significantly higher than native myeloperoxidase activities (P = 0.020; P = 0.015; P = 0.004, respectively). A statistically significant difference in total histological score was detected in the J pouch + 5% dextran sulfate sodium (DSS) group, compared to the J pouch control and the J pouch + 5% DSS + PHGG groups (P < 0.01 and P < 0.01, respectively). There was a significant overgrowth of aerobes and anaerobes in the J pouch + 5% DSS group. This study demonstrated that rectal administration of PHGG attenuates the severity of pouchitis in a rat model. In conclusion, PHGG may be an additional therapeutic strategy for the treatment of pouchitis.

The isolation and purification of biologically active recombinant and native autoantigens for the study of autoimmune disease

The expression of recombinant, biologically active mouse myeloperoxidase (MPD) and the recombinant non-collagenous (NC1) domain of mouse collagen alpha 3 Type IV was achieved for the first time in Sf21 cells ( Spodoptera frugiperda ovarian insect cells) using a baculovirus expression system. Following purification, the proteins were identified by reducing and non-reducing SDS-PAGE electrophoresis. Recombinant mouse MPO has a molecular weight of approximately 90 kDa and mouse α3(IV)NC1 approximately 32 kDa. In addition, milligram quantities of native mouse myeloperoxidase were purified from 32Dcl3 cells. Both native and recombinant myeloperoxidase were biologically active. This study also demonstrated that the immunization of myeloperoxidase deficient ( Mpo −/−) mice with purified recombinant mouse myeloperoxidase induced a significant antibody response to native myeloperoxidase.

Mechanism of interaction of betanin and indicaxanthin with human myeloperoxidase and hypochlorous acid

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils and contributes to the damage caused by these inflammatory cells. It is produced from H 2O 2 and chloride by the heme enzyme myeloperoxidase (MPO). Based on findings that betalains provide antioxidant and anti-inflammatory effects, we performed the present kinetic study on the interaction between the betalains, betanin and indicaxanthin, with the redox intermediates, compound I and compound II of MPO, and its major cytotoxic product HOCl. It is shown that both betalains are good peroxidase substrates for MPO and function as one-electron reductants of its redox intermediates, compound I and compound II. Compound I is reduced to compound II with a second-order rate constant of (1.5 ± 0.1) × 10 6 M −1 s −1 (betanin) and (1.1 ± 0.2) × 10 6 M −1 s −1 (indicaxanthin), respectively, at pH 7.0 and 25 °C. Formation of ferric (native) MPO from compound II occurs with a second-order rate constant of (1.1 ± 0.1) × 10 5 M −1 s −1 (betanin) and (2.9 ± 0.1) 10 5 M −1 s −1 (indicaxanthin), respectively. In addition, both betalains can effectively scavenge hypochlorous acid with determined rates of (1.8 ± 0.2) × 10 4 M −1 s −1 (betanin) and (7.7 ± 0.1) × 10 4 M −1 s −1 (indicaxanthin) at pH 7.0 and 25 °C. At neutral pH and depending on their concentration, both betalains can exhibit a stimulating and inhibitory effect on the chlorination activity of MPO, whereas at pH 5.0 only inhibitory effects were observed even at micromolar concentrations. These findings are discussed with respect to our knowledge of the enzymatic mechanisms of MPO.

Anti-myeloperoxidase antibodies in sera from patients with propylthiouracil-induced vasculitis might recognize restricted epitopes on myeloperoxidase molecule.

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic antibodies (ANCA) in primary systemic vasculitis. It is known that propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The production of anti-MPO antibodies in patients with PTU-induced vasculitis may be different from that in patients with primary microscopic polyangiitis (MPA). One possible reason for this may be differences in epitope recognition. The aim of this study is to compare the epitopes of antibodies to MPO in sera from patients with PTU-induced vasculitis (n = 10) and MPA (n = 10). The sera were collected and used to inhibit monoclonal antibodies against human MPO (3D8 and 6B9) and affinity purified, horseradish peroxidase conjugated human anti-MPO antibodies (Pab1-HRP, Pab2-HRP) in a competitive inhibition enzyme-linked immunosorbent assay (ELISA) system using soluble human MPO as solid phase ligand. The Pab1-HRP and Pab2-HRP were affinity purified from plasma exchanges of a patient with PTU-induced vasculitis and a patient with MPA, respectively. The inhibition rates were evaluated and compared between the PTU and primary MPA groups. In the PTU group all 10 sera could inhibit 3D8: the average inhibition rate was 44.7% +/- 5.0%; 9/10 sera could inhibit 6B9: the average inhibition rate was 35.6% +/- 6.0%. However, in the MPA group all 10 sera could inhibit 3D8 and 6B9; the average inhibition rates were 68.4% +/- 16.1% (P < 0.01) and 62.2% +/- 17.2% (P < 0.01), respectively. Sera in both the PTU and MPA groups could inhibit Pab1-HRP and the inhibition rates were 81.4% +/- 9.4%versus 86.6% +/- 17.2% (P > 0.05). However, the average inhibition rate for Pab2-HRP in the MPA group was significantly higher than that in the PTU group (76.3% +/- 7.8%versus 58.9% +/- 15.5%, P < 0.01). We conclude that anti-MPO antibodies from patients with PTU-induced vasculitis and from patients with primary MPA could recognize more than one epitope on the native MPO molecule. Although the epitopes overlapped between the two groups, the epitopes of anti-MPO antibodies from patients with PTU-induced vasculitis might be more restricted.

Oxides of Nitrogen (NO˙ and NO2–) as Cofactors of the Myeloperoxidase System

Myeloperoxidase is the main peroxisomal protein of neutrophils, monocytes, and a subpopulation of tissue macrophages; it plays the key role in protective and inflammatory responses of the organism. This role is mediated by various diffusible radicals formed during oxidative reactions catalyzed by the enzyme heme. Myeloperoxidase and nitric oxide synthase are stored in peroxisomes. Nitric oxide reacts with the heme of myeloperoxidase. Low nitric oxide concentrations increase peroxidase activity through reduction of Compound II to native myeloperoxidase. Conversely, high nitric oxide concentrations inhibit the catalytic activity of myeloperoxidase through formation of inactive nitrosyl-heme complexes. Such effect of nitric oxide on catalytic activity of myeloperoxidase has various consequences for infectious and local inflammatory processes. Another oxide of nitrogen, nitrite, is a good substrate for myeloperoxidase Compound I but slowly reacts with Compound II. Nitrogen dioxide is formed after nitrite oxidation by myeloperoxidase. Formation of nitrogen dioxide is another protective mechanism and nitration of microbial proteins by myeloperoxidase can represent an additional protective response of peroxisomes.

Anti-native and recombinant myeloperoxidase monoclonals and human autoantibodies.

Myeloperoxidase (MPO) is one of the main antigen targets of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic vasculitides. It has been suggested that anti-MPO antibodies may recognize a single epitope on recombinant MPO. If confirmed on native MPO, this might allow specific therapeutic intervention with anti-idiotypic MoAbs to prevent antibody antigen interaction which is thought to cause activation of neutrophils and vasculitis. We searched for restriction in the epitope recognition profile in 50 patients with anti-MPO autoantibodies, using both native and recombinant MPO. Mouse monoclonals were purified and tested in competition assays. At least four epitopes were identified on native MPO using these monoclonals and only two were conserved on recombinant MPO. We found that human MPO autoantibody response was not restricted to a single epitope on native MPO, as all sera tested did not show the same profile in competitive studies with monoclonals. Furthermore, 30% of human anti-native MPO sera failed to recognize rMPO.

Native and recombinant proteins to analyze auto-antibodies to myeloperoxidase in pauci-immune crescentic glomerulonephritis

The prevalence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) in pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is dependent on the assay(s) used. We investigated the frequency of MPO–ANCA as detected by different assays for MPO–ANCA in a large cohort of patients with biopsy-proven pauci-immune NCGN. Sera from 121 consecutive untreated patients presenting with pauci-immune NCGN were tested for ANCA directed to proteinase-3 (PR3) at diagnosis. PR3–ANCA negative sera were tested by direct ELISA using recombinant or native MPO and by capture ELISA using two different specific monoclonal antibodies directed to MPO and three different antigenic sources. Sera from 80 relevant disease controls were tested to explore the specificity of the different assays. Thirty-eight out of 121 patients (31%) with pauci-immune NCGN did not have PR3–ANCA. Sufficient amounts of serum from 30 of these 38 PR3–ANCA negative patients were available for further testing. Recombinant and native MPO were recognized by similar numbers of sera in a direct ELISA (recombinant MPO: 93%, native MPO: 93%) and a capture ELISA (recombinant MPO: 77–87%, native MPO: 93%). Sera of patients with PR3–ANCA positive pauci-immune NCGN and disease controls were less frequently positive for MPO–ANCA in a capture ELISA (recombinant MPO: 3–7%, native MPO: 6–7%) than in a direct ELISA (recombinant MPO: 25%, native MPO: 13%). Both direct and capture ELISA assays using either native or recombinant MPO are sensitive techniques to detect MPO–ANCA in patients with pauci-immune NCGN. A capture ELISA performs better than a direct ELISA because it combines a higher specificity with a comparable sensitivity. Recombinant MPO is a good alternative for native MPO when used as antigen in a capture ELISA, but not when used in a direct ELISA because of lower specificity in this latter assay.

Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase

The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15°C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8×107 M−1 s−1 and 1.4×107 M−1 s−1, respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2×105 M−1 s−1 and 2.7×106 M−1 s−1. Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6.6×104 M−1 s−1 at pH 7 and 15°C). Both reactions are controlled by the same ionizable group (average pKa of about 4.0) which has to be in its conjugated base form for reaction.

Reactions of pholasin with peroxidases and hypochlorous acid

The ability of myeloperoxidase (MPO) and horseradish peroxidase (HRP) to induce chemiluminescence (CL) in Pholasin (Knight Scientific, Plymouth, UK), the photoprotein of the Common Piddock Pholas dactylus, was studied. The oxidation of Pholasin by compound I or II of HRP induced an intense light emission, whereas native HRP showed only a small effect. The luminescence observed upon incubation of Pholasin with native MPO was diminished by preincubation with catalase. Considering the high instability of diluted MPO, it is concluded that traces of hydrogen peroxide in water converted MPO to its active forms, compound I and/or II, which are able to oxidize Pholasin. Indeed, the addition of hydrogen peroxide to a mixture of MPO and Pholasin induced an intense burst of light. This emission was enhanced in degree and duration in the absence of chloride. Hypochlorous acid, the reaction product of Cl − and compound I of MPO, was itself able to elicit a luminescent response in Pholasin and this luminescence was strongly inhibited by methionine and taurine. However, both of these HOCl scavengers only slightly reduced the light emission induced by MPO/H 2O 2 in both the presence or absence of chloride. Thus, hypochlorous acid produced by the MPO/H 2O 2/Cl − system, under the conditions described in this study, did not contribute to Pholasin luminescence. The Pholasin luminescence elicited by formyl-leucyl-methionyl-phenylalanine (fMLP)-stimulated neutrophils depends both on superoxide anion radicals and higher oxidation states of myeloperoxidase (but not on hypochlorous acid). This is shown by the inhibition of luminescence with superoxide dismutase and potassium cyanide, together with the lack of effect of both methionine and taurine. The luminescence response is about eight times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone.